Antibiotic af283 and production thereof



June 24, 1969 J A -rm ETAL 3,452,136

ANTIBIOTIC AFBBS AND PRODUCTION THEREOF Filed April 17, 1968 Sheet of 2 o Ov N E O 3 a (D 8 A o 2? L0 A "3 b 0 "a 9 a: E O Q 8- E E 8 i O I 2 a] (D P- I u (9 3 0 Z G g N m N Lu J m 8 J Q m 8 1 00 g 8 O- In N O 3 v NO 7 o. O r0 -N') O 8 L *8 S o o 0 5 9 (I) 0 q- N (.LNEQBd) EONVLLEWSVHL INVENTORS JOHN HENRY MARTIN JOHN NORMAN PORTER BY LESTER ALLEN MITSCH ER ATTU/P/VEY June 24, 1969 Filed April 17, 1968 EQUENCYWMJ) J- H. MARTIN L ANTIBIOTIC AF285 AND PRODUCTION THEREOF Sheet 2 of2 c'vcbocbo 9-00 u) qcu QLNEd) ENI E;

INVENTORS JOHN HENRY MARTIN JOHN NORMAN PORTER BY LESTER ALLEN MITSCHER ATTOR/VE United States Patent Office 3,452,136 Patented June 24, 1969 US. Cl. 424-118 6 Claims ABSTRACT OF THE DISCLOSURE A new antibiotic is produced, designated AF283 by cultivating a new strain of microorganism known as Streptomyces filipinensis. The new antibiotic is active against both gram-positive and gram-negative bacteria and is thus useful in inhibiting the growth of such bacteria wherever they may be found.

This application is a continuation-in-part of our copending application Serial No. 645,501, filed June 12, 1967, now abandoned which, in turn, is a continuationin-part of our application Serial No. 564,689, filed July 12, 1966, now abandoned.

This invention relates to a new antibiotic, to its production by fermentation, to methods for its recovery and concentration from crude solutions, to processes for its purification and to methods for the preparation of its salts and other derivatives.

The present invention includes within its scope the antibiotic in dilute forms, as crude concentrates and in pure crystalline forms. These novel products are active against .a variety of microorganisms including gram-positive and gram-negative bacteria. The effects of the new antibiotic on specific bacteria and mycoplasma together with its chemical and physical properties, differentiate it from previously described antibiotics.

The new antibiotic, which we have designated AF283, is formed during the cultivation under controlled conditions of a new strain of Streptomyces filipinensis. The new antibiotic-producing streptomycete was isolated from a soil sample collected in the Dominican Republic. A viable culture of the organism has been deposited with the Culture Collection Laboratory, Northern Utilization Research and Development Division, United States Department of Agriculture, Peoria, Ill., and has been added to its permanent collection. It is freely available in this repository under its Accession No. NRRL 3217.

The following is a general description of the organism S. filipinensis, NRRL 3217, based on diagnostic characteristics observed. The underscored descriptive colors and color chip designations are taken from Jacobson et al., Color Harmony Manual, 3rd ed. (1948).

Amount of growth-Moderate to good on most media; light to very light on Czapeks Solution Agar and Hickey and Treshners Agar.

Aerial mycelium .and/or en .masse spore color.Aerial mycelium whitish to yellowish white, becoming Silver Gray (3 fe) to Beige (3 ge) in sporulation zones.

Soluble pigmcnt.-In yellowish shades and-in light to moderate amounts on most media; abundant on Tomato Paste Oatmeal and Potato Dextrose Agars; none on Czapeks Solution, Hickey and Treshners and Inorganic Salts-Starch Agars.

Reverse colors-In yellow to tan to organge shades on most media.

Miscellaneous physiological reactions.Nitrates not reduced in organic nitrate broth; only partial liquefaction of gelatin after 14 days; culture is chromogenic (produces melanin) on peptone-iron agar. Carbon source utilization according to Pridham et al. [J. Bach, 56:107-114 1948)] as follows: good utilization of l-arabinose, dfructose, lactose, d-mannitol, d-melibiose, d-tr'ehalose, dxylose .and dextrose; poor to non-utilization of adonitol, dextran, i-inositol, d-melezitose, d-raffinose, l-rhamnose, salicin and sucrose.

Morphology.4pore chains produced on the aerial mycelium in the form of hooks, loops, coils and occasionally spirals of a few turns. Spores subglobose to elliptical, 0.6-0.7,u x 0.70.9 Spores ornamented with short to medium obtuse spines as determined by electron microscopy.

A comparison was made between culture NRRL 3217 and those reference cultures of streptomycetes having the combination of: grayish spores, spiny spores, spiralled spore chains, and production of melanin on peptone-iron agar. The new isolate most closely corresponds to the strains of Streptomyces filipinensis. When it was then compared with the original description of that species, as described in The Actinomycetes, vol. II, Waksman, S. A., The Williams & Wilkins Co., Baltimore (1961), an equally close similarity was found. Therefore, in view of the lack of any significant divergence from the specific concept for S. filipinensis, culture NRRL 3217 will hereafter be considered a strain of that species.

A critical examination of the cultural, physiological and morphological features of the organism was made when it was grown on several media; including those recommended by Pridham et al. [A Selection of Media for Maintenance and Taxonomic Study of Streptomyces, Antibiotics Annual (1956-1957), pp. 947-953]. Detailed observations are recorded in Tables I, II, III, and IV below. Underscored: descriptive colors are taken from the Color Harmony Manual.

TABLE I.CULTURAL CHARACTERISTICS OF STREPTOMYCES FILIPINENSIS NRRL 3217 (Incubation: 14 days; Temperature: 28 0.]

Amount of Medium growth Aerial mycelium and/or spores Soluble pigment Reverse color Remarks Czapeks Solution Agar Very light Aerial mycelium white, thin. None Whltish Splorulation grayish, very 1g Asparagine Dextrose Agar Moderate. Aerial mycelium yellowish Yellowish; light Maize (2 hb) white, becoming Silver Gray (3 fe) in sporulation zones. sporulation light. Tomato Paste Agar do Aerial mycelium yellowish None Lt. tan (3 go) white, becoming Silver Gray (3 fe) in sporulation zones. Sporulation moderate. Hickey & Tresners Agar Light Aerial mycelium yellowish do Lt. tan (3 go) white, becoming Silver Gray l n (3 ie) to Beige (3 ge) in sporulation zones. Sporulation light.

TABLE I.CULTURAL CHARACTERISTICS OF STREPTOMYCES FILIPINENSIS NRRL 3217-Continued Amount of Medium growth Aerial mycelium and/or spores Soluble pigment Reverse color Remarks Yeast Extract Agar Moderate- Aerial mycelium whitish, Yellowish; light Yellow maple (3 ng)...

becoming Silver Gray (3 to) to Beige (3 ge) in sporulation zones. Sporulation good. Oatilake Agar Good Aerial mycelium whitish, Yellowish; moderate. Lt. wheat (2 ea) becoming Silver Gray (3 ie) to Beige (3 ge) in sporulation zones. Sporulation good.

Carvajals Oatmeal Agar .do Aerial mycelium yellowish Yellowish; light Yellow maple (3 ng) Colony surface white, becoming Silver Gray cracked and (3 fe) in sporulation zones. wrinkled. Sporulation heavy.

Tomato Paste Oatmeal Agar ..do Aerial mycelium yellowish Yellowish; abundant Amber (31c) white, becoming Silver Gray (3 is) to Beige (3 ge) in sporulation zones. Sporulation good.

Potato Dextrose Agar do Aerial mycelium yellowish do Maple (41c) white, becoming Silver Gray (3 to) in sporulation areas. Sporulation good.

Bennetts Agar Moderate- Aeria mycelium whitish, Yellowish; light Yellow maple (3 ng) becoming Silver Gray (3 fe) to Beige (3 ge) in sporulating areas. Sporulation good.

Inorganic Salts Starch Agar do Aerial mycelium whitish, None Light wheat (2 ea) becoming Silver Gray (3 to) to Beige (3 go) in sporulating areas. Sporulation good.

TABLE II.MICROMORPHOLO GY OF STREPTOMYCES FILIPINENSIS NRRL 3217 Medium Aerial mycelium and/or sporiierous structures Spore shape Spore size Spore surface Oatflake Agar- Spore chains produced on the aerial mycelium Spores subglobose 0.6-0.7 x O.70.9p Spores ornamented with short to medium in the form of hooks, loops, coils and occato elliptical. obtuse spines (as determined by sionally spirals of a few turns. electron microscopy).

TABLE IIL-MISOELLANEOUS PHYSIOLOGICAL REACTION OF STREPTOMYCES FILIPINENSIS NRRL 3217 [Temperature 28 0.]

Incubation Amount of Medium period growth Physiological reaction Organic nitrate broth. 7 days Moderate No nitrate reduction.

Do 14 days. Good Do. Gelati 7 days Moderate- No gelatin liquefaction.

14 days Good..- Partial liquefaction. Peptone on agar 24 hours do Chromogenic (melanin produced).

Table IV.Carbon source utilization pattern of Strepsuch as X-radiation, ultraviolet radiation, nitrogen mustomyces filipinensz's NRRL 3217 tard, phage exposure and the like.

[Incubationz 10 days. Temperature: 28] The fermentatlon Precess Cultivation of the organism S filipinensis NRRL 3217 1 n c I n I c xg jgg Unhzatlono may be carried out in a wide variety of liquid culture 3 media. Media which are useful for the production of the s 1 arabmo 6 novel antibiotic include an assimilable source of carbon ggiggg 2 such as starch, sugar, molasses, glycerol, etc., an assimi- Hnositol lable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc., and inorganic anions and cations, such as potassium, sodium, calcium, sulfate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc. are supplied as impurities of other constituents of the media.

Lactose d-Mannitol d-Mclezitose d-Mclibiose ifigfif; Aeration in tanks and bottles is provided by forcing sterile Salicin 1 air throughor onto the surface of the fermenting medium. Sucrose Further agitation in tanks is provided by a mechanical d Treha1ose mpeller. An antifoammg agent, such as 1% octadccanol, d Xylose 3 111 lard 011 may be added as needed. Dextrose l 3 Inoculum preparation Negatwe control O Shaker flask inoculum of S. filipinensis is prepared by 1 3, qq utilization; fail utilization; D utilization; inoculating 100 milliliters of sterile liquid medium in 500 no umlzation milliliter flasks with scrapings or washings of spores from It is to be understood that for the production of the an agar slant of the culture. The following medium is new antibiotic the present invention is not limited to this ordinarily used. particular organism only, nor to organisms fully answer- Grams ing the above growth and microscopic characteristics Molasses 20 which are given for illustrative purposes. In fact, it is Glucose 10 desired and intended to include the use of mutants Bactopeptone ..F 5

produced from the described organism by various means, Water to 1,000 milliliters.

The flasks are incubated at a temperature from 25-29 C., preferably 28 C., and agitated vigorously on a rotary shaker for 30 to 48 hours. These 100 milliliters of inocula are used to inoculate 1 liter and 12 liter batches of the same medium in 2 liter and 20 liter glass fermentors. The inoculum mash is aerated with sterile air while growth is continued for 30 to 48 hours. These batches of inocula are used to inoculate tank fermentors.

Tank fermentation For the production of the antibiotic in tank fermentors the following fermentation medium is preferably used.

Grams Soy bean flour 40 Cerelose 20 Calcium carbonate 2 Water to 1,000 milliters.

Each tank is inoculated with 3 to of inoculum made as described above. Aeration is supplied at the rate of 0.5-1.0 liter of sterile air per liter of broth per minute and the fermenting mixture is agitated by an impeller driven at 200-400 r.p.m. The temperature is maintained at 25-29 0., usually at 28 C. The fermentation is ordinarily continued for 65-90 hours at which time the mash is harvested.

Isolation procedure After the fermentation is completed, the fermented mash containing the antibiotic is filtered, preferably at about pH 2.5, to remove the mycelium. Diatomaceous earth or any other conventional inert filtration aid may be used to assist the filtration. Ordinarily the mycelial cake is washed with water and the wash is pooled with the filtrate. Optionally, the mycelial pad may be eluted with 70% aqueous methanol, the eluate filtered and concentrated to an aqueous phase. This concentrate may then be added into the beer filtrate of a subsequent fermentation to enhance the antibiotic activity available for recovery. Thereafter, the antibiotic may be recovered from the combined filtrate and wash using conventional techniques, as for example, -by adsorption on activated carbon at about pH 6.5 and elution with acidic 75% acetone. The antibioticcontaining eluate is adjusted to about pH 3.0 to 4.0 and concentrated under reduced pressure to an aqueous phase having a volume slightly larger than one-hundredth of the original mash volume. The concentrate is adjusted to a pH of 6.0-6.5. The antibiotic in the concentrate is further purified by passing the concentrate through a weakly acidic cation exchange resin. The antibiotic which is retained on the resin is eluted and the eluate is concentrated and lyophilized to yield the semi-purified antibiotic.

Purification procedure Futher purification of the antibiotic activity and its separation into two components, termed AF283a and AF283 may be effected by partition chromatography on diatomaceous earth or Celite or by displacement chromatography on CM Sephadex C-25 cation exchanger.

In the partition chromatography method, the semipurified material is charged onto a column packed with acid-washed Celite which is wetted with the aqueous phase of a well-shaken mixture of 5 parts of sec-butanol and 4 parts of water. The column is then developed with the organic phase of this system to elute the desired a and 5 components separately as indicated by optical density readings at 270 my. Fractions containing the a component are combined, desolventized and lyophilized to yield purified AF283oz. In a similar manner fractions containing the other portion of the antibiotic activity, the 5 com ponent, are combined, desolventized and lyophilized to yield purified AF283B.

In the displacement chromatography method the semipurified antibiotic is charged onto a column packed with CM Sephadex which is previously treated with 0.1 N acetic acid for 48 hours, washed and equilibrated with 3% ammonium formate at pH 7.5. The charged column is then developed using addition 3% ammonium formate at pH 7.5. The eluate is monitored with ultraviolet absorption readings at about 254 mg and appropriate fractions containing the 0: component are collected separately from fractions containing the ,8 component. Both the a component and the 18 component may be recovered by repeated lyophilization of the respective eluates or, optionally, by adsorption on carbon followed by elution with aqueous acetone and subsequent lyophilization of the eluates.

The novel antibiotics of this invention are basic substances and in their free base form contain the elements carbon, hydrogen, oxygen, nitrogen in substantially the following percentages by weight (it will be noted that the wcomponent is quite hygroscopic as to free base and the amount of moisture remaining after drying aifects the microanalytical figures which follow).

Alpha component Mild drying Thorough drying Beta component Carbon Hydrogen Oxygen (direct) itrogen In this context, mild drying conditions consist of heating for three hours at 78 C. under reduced pressure in the presence of P 0 Extensive drying consists of heating for 16 hours at 100 C. under reduced pressure in the presence of P 0 The following are various physical characteristics of the beta component:

Neutralization equivalent of the hydrochloride salt 864.

The optical rotation is [a] =28.6 (i3) (c.=0.874 in 0.01 N HCl).

Ultraviolet maxima occur at:

264 m (Elg... =255) in 0.1 N HCl 290 m (E}'Z;'.,, =315) in 0.1 N NaOH 264 m, (E}'T;',,, =275) in methanol An infrared absorption spectrum in a KBr pellet is prepared in a standard manner. It exhibits characteristic absorption in the infrared region of the spectrum at the following wavelengths expressed in microns: 3.00, 3.43, 6.10, 6.32, 6.65, 6.95, 7.20, 7.43, 7.98, 855,897, 9.52, 12.20, 13.25, 14.40. The infrared curve of the beta component is shown in FIGURE 2 of the accompanying drawings.

The antibiotic has no definitive melting point. On heating, it turns to a light tan at 134 C. and gradually darkens as the temperature is increased.

The beta component shows the following R, values in the solvent systems indicated below using bioautographs against Corynebacterium xerosis as the detection organrsm:

R i value Solvent system 0.20 3% ammonium chloride (aqueous). 0.58 m-(aesgl saturated with water with 2% perfluorobutyric acid a e 0.01.- chloroform, 10 parts; pyridine, 4 parts; acetic acid, 4 parts;

Water, 5 parts. 0.01... Sec-butanol, 3 parts; pyridine, 6 parts; s-collidine, 6 parts;

water, 3 arts. 0.80 phenol, 1 part; 2% dichloroacetic acid, 1 part.

The beta component, when spotted on paper, gives the following results:

Spray reagent: Color found Ninhydrin (0.25%) Dark grey. p-Nitrobenzene-diazonium fluoroboborate (1%) Yellow.

An infrared absorption spectrum of the alpha component in a KBr pellet is prepared in a standard manner. It exhibits characteristic absorption in the infrared region of the spectrum at the following wavelengths expressed in microns: 3.10, 3.28, 3.43, 6.02, 6.25, 6.70, 6.95, 7.28, 7.44, 7.95, 9.00, 9.50, 12.20, 13.23, 14.30. The infrared curve of the alpha component is shown in FIGURE 1 of the accompanying drawings.

The alpha component shows the following R; values in the solvent system indicated below using bioautographs against Corynebacterium xerosis as the detection orgamsm:

R, value: Solvent system 0.32 ammonium chloride.

0.65 90% phenolzwater plus 2% dichloroacetic acid (added to bottom phase).

0.25 m-Creso1:90% phenol:0.2M

morpholine:0.2M acetic acid (5 :5 27:3).

0.24 m-Cresol saturated with water plus 2% heptafluorobutyric acid (added to lower phase). 0.24 Sec.-butano1:acetic acid:water The novel antibiotic is active in vivo against a variety of gram-positive microorganisms, such as staphylococci and diplococci. The new antibiotic is thereby potentially useful as a therapeutic agent in treating bacterial infections in mammals caused by such microorganisms. The new antibiotic can be expected to be usefully employed for controlling such infections by topical application or parenteral administration or when taken orally.

The usefulness of the new antibiotic is demonstrated by its ability to control systemic lethal infections in mice. AF283 shows high in vivo antibacterial activity in mice against Staphylococcus aureus, strain Smith, Staphylococcus aureus, strain Rose, and Diplocoscus pneumonia, SVL, when administered by a single dose to groups of Carworth Farms CF-l female mice, weight about 20 grams infected intraperitoneally with a lethal dose of these bacteria in trypticase soy broth (TSP) dilutions of a five-hour TSP blood culture.

Table V, following, illustrates the in vivo antibacterial activity of AF283/8.

TABLE V.IN VIVO ANTIBACTERIAL ACTIVITY OF AF283B [5-6 Days Postinfection] Survivors/total Mg./kg. of

vt. S.O.D.

Staphylococcus aureus Strain Smith (Gone. 10

Staphylococcus aureus Strain Rose (Gone. 10").

Diplococcus pneumoniae SVI (Gone. 10*).

l S.O.D.=single oral dose. 2 S.S.C.=single subcutaneous dose.

Two groups of controls were used: 20/20 and 48/50 of the infected non-treated controls died within one day.

TABLE VL-IN VIVO ANTIBACTERIAL ACTIVITY OF AF283c:

[5-6 Days Postiniection] MgJkg. Survivors] body wt. total S.S.C

Staphylococcus aurcus Smith strain. 5/5 20 10/10 5 10/10 1. 2 5/10 0. 3 1/10 1 S.S.C.=single subcutaneous dose.

The new antibiotic possesses both in vivo and in vitro activity against Mycoplasma gallisepticum. Table VII below shows the in vitro activity of AF283 obtained against this organism.

TABLE VIL-IN VITRO ACTIVITY OF AF283 AGAINST 111. GALLISEP TIC U111 {Broth Bilution Test] 1 MiniAum inhibitory concentration (meg/m1.)

Test compound A B 1 Plated out after 3, 7, and 14 days and checked for colonies after an additional 4 days incubation.

A= Turkey strain; B S6 strain from chickens.

Table VIII shows the activity of AF283 against M. gallisepticum in one-week old chicks.

TABLE VII I.IN VIV(I)NACTIVITY OF F283 AGAINST M. GALLISEPTIC U111 ONE-WEEK OLD CHICKS Avg. bird weights Dose, (days postiniection) Survivors mg. Test compound bird 0 7 14 21 S/T 1 Percent S AF383. 6. 25 68. 6 117. 5 172. 6 255. 5 19/20 AF283- 1. 56 69. 2 114. 6 159. 7 228. 2 19/20 95 A1283. 0. 39 68. 4 114. 2 153.6 222. 3 19/20 95 Infected (untreated) 69. 5 96. 7 117. 8 7. 2 12/20 60 Uninfected (untreated) 70. 2 124. 4 194. 7 293. 8

1 S/T Survivors to total tested.

Twenty chicks per group, infection administered via air sac; single treatment at time of infection subcutaneously. The above results against M. gallisepticum indicates a potentially important commercial use of the antibiotic in the cure or prevention of certain diseases affecting poul- When tested in vitro at a concentration of 25 /1111 the antibiotic was found to have pronounced antibacterial activity against Corynebacterium xerosis, NRRL B-l397 and slight activity against Mycobacterium smegmatis, No. 607 Stansly R-99 and Staphylococcus aureus, strain Smith.

The invention will be described in greater detail in conjunction with the following specific examples.

Example 1.Inoculum preparation A typical medium used to grow the primary inoculum is prepared according to the following formula:

Grams Mollasses 20 Glucose 10 Bactopeptone Water to 1,000 milliliters.

Example 2.Fermentation A fermentation medium is prepared according to the following formula:

Grams Soy bean flour 40 Crelose 20 Calcium carbonate 2 Water to 1,000 milliliters.

The fermentation medium is sterilized at 120 C. with steam at 15 pounds pressure for 45-60 minutes. The pH of the medium after sterilization is 6.4. Three hundred liters of sterile medium in a 400 liter tank fermentor is inoculated with 12 liters of inoculum such as described in Example 1, and the fermentation is carried out at 28 C. using Hodag LG-8 oil as a defoaming agent. Aeration is supplied at the rate of 0.5 liter of sterile air per liter of mash per minute. The mash is agitated by an impeller driven at 300 revolutions per minute. At the end of approximately 70 hours of fermentation time the mash is harvested.

Example 3 .Isolation Three hundred and thirty liters of the fermented mash from Example 2 is adjusted to pH 2.5 with concentrated hydrochloric acid and diatomaceous earth added in the portion of 3% Weight per volume. The broth is filtered, the filter cake washed with 50 liters of water, and the filtrate and wash combined. The combined filtrate and washings are adjusted to pH 6.5 with sodium hydroxide and 1200 grams of Darco G-60 and 1200 grams of diatomaceous earth are added and the mixture stirred for 30 minutes and then filtered. The carbon pad is washed with water and the filtrate and washings are discarded. The antibiotic-rich carbon pad is sluriied with liters of 75% aqueous acetone and the slurry poured into a glass column having nine-inch diameter. The carbon column is eluted with a mixture of acetone, water and 2 N hydrochloric acid in the proportion of 75 :22:3 by volume. Seven separate 9 liter fractions are collected and each fraction assayed for antibiotic activity using C. xerosis as the test organism. Fractions 4, 5, 6 and 7, containing the major portion of the eluted activity are combined. The

combined fractions are adjusted to pH 3.5 with sodium hydroxide and concentrated under reduced pressure to an aqueous phase of approximately 4 liters.

The aqueous concentrate is adjusted to pH 6.0 to 6.5 with sodium hydroxide and further purified by ion exchange chromatography. A quantity of IRC-SO resin is washed with hydrochloric acid until the pH remains at 1.5 on standing. The resin is then washed with several portions of water (decanting after each Wash) and then filtered on a Buchner funnel and washed with distilled water. The washed resin is converted into the ammonium ion form by adding concentrated ammonia until a pH between 8.3 to 9.0 is reached. The treated resin is poured into a tall glass column mm. diameter) to a height of 45 cm.. The column is Washed with about 9 liters of distilled water. A layer (2 to 3 cm.) of small glass heads is used to cover the resin surface. The column is not allowed to run dry.

The resin column, prepared as described above, is charged with the concentrate of the Darco G-60 eluate. The flow of the column is adjusted to a rate of about 30 mlfof liquid per 'minute. When all of the charge is on the column, distilled Water is added and the flow rate is increased to about ml. of liquid per minute. The first fraction of eluate saved is taken from the point of the first appearance of a black to dark brown color and continued to the point where the eluate is essentially colorless. When this point is reached 1 liter of 0.5% ammonium hydroxide (16.7 ml. of concentrated reagent grade ammonium hydroxide per liter of solution) is added to the column and the flow rate reduced to 40 ml. of liquid per minute. When all of the 0.5% ammonium hydroxide is on the column, 20% ammonium hydroxide is used and the elution is continued until a dark or warm zone is about 15 cm. from the bottom of the resin column marking the end of the second fraction. Elution of the column is continued collecting separate 20' ml. fractions. Optical density readings at 270 III/L are noted on dilutions (.20 ml. to 10.0 ml.) from every fifth fraction. Readings are obtained as given in the table below.

TABLE IX.OPIIOAL DENSITY OF IRC-50 COLUMN ELUATE FRACTIONS Fraction No. O.D. Fraction No. O.D. Fraction No. O.D.

Fractions 61 through (700 ml.) are combined immediately. About 500 ml. of n-butanol is added and the mixture is concentrated to an aqueous phase of about 200 ml., pH 8.4 to 8.7. The concentrate is then shell frozen and lyophilized to yield 5.5 g. of whitish to light tancolored, semi-purified antibiotic.

Example 4.Purification The semi-purified antibiotic from Example 3 is further purified and separated into two components, termed AF283a and AF2835, by either of the following two procedures: partition column chromatography on Celite or displacement chromatography using CM Sephadex cation exchanger.

TABLE X.OPTICAL DENSITY OF OELITE COLUMN ELUATE FRACTIONS Fraction No. 0.1). Fraction No. CD. Fraction No. 0.1).

Fractions 50 through 70 are combined, desolventized, and then lyophilized to yield 146 mg. of AF283u, a white fluffy solid material. Similarly, fractions 85 through 125 are combined, desolventized and then lyophilized to yield 374 mg. of AF283 8, likewise, a white fiuffy solid.

Method B.In the displacement method using CM Sephadex cation exchanger, about 100 g. of CM Saphadex C-25 medium is allowed to swell in 0.1 N acetic acid (800 ml.) for 48 hours. The excess acid is decanted and the swollen exchanger washed batchwise several times with 3% ammonium formate adjusted to pH 7.5 with ammonium hydroxide. The cation exchanger is then packed into a 2-inch diameter glass column to a height of 6.5 inches. An additional 9 l. of ammonium formate is passed through the column to ensure complete equilibration. About 6.0 grams of AF283, obtained from Example 3, is stirred into a suitable quantity of ammonium formate and the charge is then percolated into the prepared column. The charged column is then developed with ammonium formate, the eluate being monitored by a Uvicord Control Unit, type 4701A, set to read ultraviolet absorption at about 254 m The fraction containing AF2830 (volume about 1.5 l.) is collected and kept separate from the fraction containing AF2835 (volume about 7.5 1.). AFZSSm and AF28318 are recovered from the two fractions as follows: The ammonium formate solution of the 6 component is passed through a column containing a mixture of 22 g. of Norit A and 22 g. of acid washed Celite. The charged column is Washed with 1.6 to 2.0 l. of water to insure removal of trapped ammonium formate. AF28313 is eluted from the column with about 800 ml. of 50% aqueous acetone. The eluate is concentrated under reduced pressure to an aqueous phase and lyophilized. A yield of 765 mg. of purified AF283B is obtained. The eluate containing the a component is treated in a similar manner using appropriately smaller quantities of Norit A, Celite, water, and aqueous acetone. A yield of about 80 mg. of purified AF283u is obtained.

Alternately, in other experiments the antibiotic is isolated in solid form essentially free from ammonium formate by repeated lyophilization.

The chemical analysis of each of the alpha and beta components of AF283 and the other physical and biological properties of the new antibiotics have already been described.

Example 5.-Preparation of the hydrochloride salt of AF2833 AF28318, 174 mg., is dissolved in 5 ml. of water and the solution adjusted to pH 1.3 with 6 N hydrochloric acid. Acetone is added until the solution just turns cloudy. The cloudy mixture is filtered through a sintercd glass funnel. Additional acetone, 30 ml., is added to the filtrate and the mixture placed in the chillroom for several hours.

The cold solution is filtered through a sintered glass funnel and a fiocculent white precipitate is obtained which is washed first with acetone and then with diethyl ether. The washed salt weighs 111 mg. after drying at room temperature over P 0 under reduced pressure.

AF283 8.HC1 contains the following elements in substantially the following percentages by weight:

Carbon 45.43 Hydrogen 6.30 Nitrogen 13.63 Chlorine 12.72,12.69 Oxygen (direct) 21.21

The acid salt has a neutralization equivalent of 864, pKa 3.5.

Ultraviolet maxima occur at:

264 mu (El g =230) in 0.01 N H01 290 mu (E} =275) in 0.01 N NaOH The salt (prepared in KBr pellet) exhibits characteristic absorption in the infrared region of the spectrum at the following wavelengths expressed in microns: 2.95, 3.03, 6.01, 6.58, 6.91, 7.33, 7.94, 8.10, 8.50, 8.80, 8.91, 9.46, 12.15.

Example 6.Preparation of the sulfate salt of AF283B AF283B, mg., is dissolved in 1 ml. of water and the solution filtered through a sintered glass funnel. Two drops of water are used as a wash. The combined filtrate and washings are adjusted to pH 2.0 with 3 N sulfuric acid. Isopropanol is added to the acidified solution dropwise until a precipitate forms (about 3 drops). The suspension is placed in a chillroom for 2 hours and then filtered. The recovered white precipitate is washed with isopropanol and dried at room temperature over P 0 under reduced pressure.

Example 7.Preparation of the helianthate salt of AF283B Carbon 53.55 Hydrogen 5.88 Nitrogen 14.52 Sulfur 5.33 Oxygen (by difi.) 20.72

Ultraviolet and visible maxima occur at:

272 m (Ei'Z,.,,=273) in 0.01 N H01 The salt (prepared in KBr pellet) exhibits characteristic absorption in the infrared region of the spectrum at the following wavelength expressed in microns: 3.00, 6.01, 6.25, 6.58, 6.92, 7.05, 7.32, 7.61, 8.25, 8.58, 8.80, 8.96, 9.40, 9.70, 9.92, 10.35, 11.78, 12.16, 13.32, 14.35.

530 mg. of AF283 8 are warmed in 15 ml. of distilled water and the solution filtered through a sintered glass funnel. 9.13 mg. of reineckate salt is warmed in 22 ml. of deionized water and this solution filtered through a sintered glass funnel. Both solutions are warmed to about 50 C. separately and then mixed together. The mixture is allowed to come to room temperature slowly. The reineckate salt of the antibiotic is recovered by filtration using a sintered glass funnel and washed with three separate 3 ml. portions of cold water. The washed salt is dried at room temperature over P under reduced pressure: yield 657 mg.

The'reineckate salt of the antibiotic contains the following elements in substantially the following percentages by weight:

Carbon 33.34 Hydrogen 4.85 Nitrogen 19.71 Sulfur 17.78 Chromium 8.17 Ox'ygen (by diff.) 16.15

Ultraviolet maxima occur at:

304 mu (E}?; =270) in water The salt (prepared in KBr pellet) exhibits characteristic absorption in the infrared region of the spectrum at the following wavelengths expressed in microns: 2.96, 4.72, 6.00, 6.18, 6.62, 6.91, 7.10, 7.90, 8.85, 9.43, 12.10.

Example 9.-Preparation of the benzoyl derivative of AF28318 AF283fl (174 mg), sodium bicarbonate (650 mg.), water (12 m1.) are added to a test tube and shaken. The resulting mixture is filtered through a sintered glass funnel to remove a small amount of insolubles. Benzoyl chloride (0.4 ml.) is added to the filtrate and the mixture is shaken vigorously for 15 minutes, cooled and filtered through a sintered glass funnel. The precipitate is washed with cold water and dried over P 0 under reduced pressure. The dried reaction product is washed several times with ether on a funnel. The yield of the benzoyl derivative of AF283B is 171 mg.

The novel benzoyl derivative of the antibiotic contains the following elements in substantially the following percentages by weight:

Carbon 59.14 Hydrogen 5.76 Nitrogen 11.66 Oxygen (direct) 20.84

Ultraviolet maxima occurs at 293 m (Elzfm.=235) in 0.01 N NaOH The derivative (prepared in KBr pellet) exhibits characteristic absorption in the infrared region of the spectrum at the following wavelengths expressed in microns: 2.93, 3.35, 6.05, 6.30, 6.58, 6.70, 6.92, 7.15, 7.60, 7.86, 8.20, 8.50, 8.94, 9.35, 9.70, 12.15, 14.00.

Example 10.-Preparation of the p-iodobenzoyl derivative of AF283fi pressure. The dried precipitate, weight 454 mg., is triturated with 20 ml. of warm benzene (70 C.) and filtered. The benzene insoluble precipitate is washed with petroleum ether and air dried. The yield of the p-iodobenzoyl derivative of AF283,8 is 213 mg.

The p-iodobenzoyl derivative of AF283fi contains the following elements in substantially the following percentages by weight:

Carbon 42.62 Hydrogen 3.74 Nitrogen 7.42 Iodine 29.41 Oxygen (by diff.) 16.81

The derivative (prepared in KBr pellet) exhibits characteristic absorption in the infrared region of the spectrum at the following wavelengths expressed in microns: 2.93, 5.76', 6.22, 6.30, 6.58, 6.75, 7.19, 7.93, 8.23, 8.52, 8.98, 9.30, 9.92, 11.40, 11.87, 13.35.

Example 11.Preparation of p-bromobenzoyl derivative of AF283 3 AF283/3 mg.), sodium bicarbonate (650 mg), p-bromobenzoyl chloride (500 mg.), dioxane (5 ml.), and water (5 ml.) are added to a test tube. The resulting mixture is shaken vigorously for 20 minutes after adjusting the pH of the mixture to 10.0 with 1 N sodium hydroxide. A white precipitate is obtained on filtering. The precipitate is washed with water and dried over P 0 under reduced pressure. The dried precipitate, weight 215 mg., is suspended in 10 ml. of benzene, heated on a steam cone for a few minutes and filtered. The benzene insoluble precipitate is washed with petroleum ether and air dried. The yield of the p-bromobenzoyl derivative of AF283B is 159 mg.

The novel p-bromobenzoyl derivative of AF283fl contains the following elements in substantially the following percentages by weight:

Ultraviolet maxima occur at:

286 my. (E%" =183) in 0.01 N NaOH The derivative (prepared in KBr pellet) exhibits characteristic absorption in the infrared region of the spectrum at the following wavelengths expressed in microns: 2.91, 5.76, 6.02, 6.29, 6.67, 6.73, 6.95, 7.15, 7.23, 7.90, 8.52, 8.97, 9.88, 11.38, 11.81, 13.30.

Example 12.-Preparation of hydrobromide salt of AF283/3 AF283B, 100 mg, is dissolved in 1 ml. of water and the solution filtered through a sintered glass funnel to remove a small amount of insolubles. The filtrate is adjusted to pH 1.8 with 48% hydrobromic acid. Tetrahydrofuran is added and the cloudy mixture is placed in the chillroom for several hours after which time a solid forms on the walls of the flask. The spent liquid is decanted and the solid triturated with fresh tetrahydrofuran and scraped free from the walls of the flask. The solid is collected on a sintered glass funnel, and washed with diethyl ether and dried: yield 75 mg.

The novel AF283p hydrobromide contains the following elements in substantially the following percentages by weight:

Oxygen (direct) 17.23

Ultraviolet maxima occur at:

264 m (El"m =213) in 0.01 N H01 Corresponding hydrochloride, hydrobromide, etc., salts of the alpha component may be prepared in an exactly analogous manner. Analytical data are included below.

Analysis of hydrobromide salt: C, 42.12; H, 5.63; N, 9.30; Br, 22.63; 0, 18.60.

Analysis of hydrochloride salt: C, 49.53; H, 6.07; N, 9.93; Cl, 11.52, 0, 21.92.

What is claimed is:

1. A substance effective in inhibiting the growth of gram-positive and gram-negative bacteria selected from the group consisting of antibiotic AF283B which in its free base form is characterized as follows:

Analysis.--Carbon, 50.28%; hydrogen, 6.77%; oxygen (direct), 26.09%; nitrogen, 15.32%; optical rotation: [a] =-28.6 (:3) (c.=0.874 in 0.01 N HCl; ultraviolet maxima:

264 mp (E}f'.,,=255) in 0.1 N H01 290 my. (E}Z;, =315) in 0.1 N NaOH 264 m (E}?;,, =275) in methanol infrared spectrum: as shown in FIGURE 2.

3. A substance effective in inhibiting the growth of gram-positive and gram-negative bacteria selected from the group consisting of antibiotic AF283u which in free base form is characterized as follows:

P ercent Mild drying Extensive drying Analysis conditions conditions Carbon 51. 08 53.15 Hydrogen 7. 59 6. 34 Oxygen (direct) 27. 88 26. 35 Nitrogen 11. 83 ll. 33

16 optical rotation: [a] =--6.1 (12.3) (c.=1.3l0 in 0.1 N HCl); ultraviolet maxima:

Mild drying Extensive drying Analysis conditions conditions Carbon ,51. 08 53.15 Hydrogen 7. 59 6. 34 Oxygen (direct)- 27. 88 26. 35 Nitrogen 11.83 11. 33

optical rotation: [a] =6.1 (L -2.3) (c.=l.310 in 0.1 N HCl); ultraviolet maxima:

infrared spectrum: as shown in FIGURE 1.

5. A compound selected from the group consisting of antibiotic AF283B as characterized in claim 2 and antibiotic AF283 as characterized in claim 4.

6. A process which comprises cultivating Streptomyces filipinensis NRRL 3217 in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic conditions for a period of from to 90 hours and at a temperature of from 25 to 29 C. until substantial antibiotic activity is imparted to said medium by the production of a compound as characterized in claim 5, and is separated into two components AF283B and AF283a by partition column chromatgraphy.

References Cited Miller, The Pfizer Handbook of Microbial Metabolites, McGraw-Hill Book Company, Inc., New York, pages 126 and 127, January 1962.

ALBERT T. MYERS, Primary Examiner.

JEROME D. GOLDBERG, Assistant Examiner.

US. Cl. X.R. 

